**Annex 15. Item 5.1. – Chapter 3.8.12. Sheep pox and goat pox**

###### 2	MEETING OF THE BIOLOGICAL STANDARDS COMMISSION

3	**Paris, 4–8 September 2023**

4

5	**CHAP T E R  3 . 8 . 1 2 .**

# 6	S H E EP P OX A N D GOA T POX

7	**SUMMARY**

8 *Sheep pox and goat pox are contagious, viral diseases of sheep and goats characterised by fever,*

9 *generalised papules or nodules, vesicles*(*rarely*), *internal lesions*(*particularly in the lungs*), *and death. Both*

10 *diseases are caused by strains of capripoxvirus, all of which can infect sheep and goats. Although most of*

11 *the strains examined cause more severe clinical disease in either sheep or goats, some strains have been*

12 *isolated that are equally pathogenic in both species.*

13 *Sheeppox virus*(*SPPV*) *and goatpox virus*(*GTPV*) *are the causative agents of sheep pox and goat pox, and*

14 *with lumpy skin disease virus*(*LSDV*)*make up the genus*Capripoxvirus *in the family*Poxviridae*. Sheep pox*

15 *and goat pox are endemic in Africa north of the Equator, the Middle East and Asia, while some parts of*

16 *Europe have experienced outbreaks recently. See WAHIS*(*https://wahis.woah.org/#/home*) *for recent*

17 *information on distribution at the country level. ~~Countries that reported outbreaks of the disease between~~*

18 *~~2010 and 2015 include Bulgaria, Chinese Taipei, Israel, Kazakhstan, Kyrgyzstan, Mongolia, Morocco,~~*

19 *~~Greece and Russia, with Greece, Israel and Russia having experienced recurring incidences.~~*

20 ***Identification of the agent:****Laboratory confirmation of capripoxvirus is most rapid using the polymerase*

21 *chain reaction*(*PCR*) *method in combination with a clinical history consistent with generalised capripoxvirus*

22 *infection. Isolation of the virus is possible as capripoxviruses will grow on tissue culture of ovine, caprine or*

23 *bovine origin, although field isolates may require up to 14 days to grow or require one or more additional*

24 *tissue culture passage*(*s*)*. The virus causes intracytoplasmic inclusions that can be clearly seen using*

25 *haematoxylin and eosin staining. The antigen can also be detected in tissue culture using specific sera and*

26 *immunoperoxidase or immunofluorescence techniques. Capripoxvirus antigen and inclusion bodies may be*

27 *seen in stained cryostat or paraffin sections of biopsy or post-mortem lesion material.*

28 *~~An antigen-detection enzyme-linked immunosorbent assay~~*~~(*ELISA*) *using a polyclonal detection serum*~~

29 *~~raised against a recombinant immunodominant antigen of capripoxvirus has been developed.~~*

30 ***Serological tests:****The virus neutralisation test is the most specific serological test. The indirect*

31 *immunofluorescence test is less specific due to cross-reactions with antibody to other poxviruses. Western*

32 *blotting using the reaction between the P32 antigen of capripoxvirus with test sera is both sensitive and*

33 *specific, but is expensive and difficult to carry out. An enzyme-linked immunosorbent assay*(*ELISA*) *has*

34 *been developed and validated to detect antibodies to capripoxviruses, however it cannot differentiate*

35 *between SPPV, GTPV and LSDV.*

36 *~~The use of this antigen, or other appropriate antigens expressed by a suitable vector, in an ELISA offers the~~*

37 *~~prospect of an acceptable and standardised serological test in the future.~~*

38 ***Requirements for vaccines:****Live and inactivated vaccines have been used for the control of*

39 *capripoxviruses. All strains of capripoxvirus so far examined share a major neutralisation site and some will*

40 *cross protect. Inactivated vaccines give, at best, only short-term immunity.*

## 41	A. INTRODUCTION

42 The *Capripoxvirus*genus, in the family *Poxviridae,*consists of three species – lumpy skin disease virus (LSDV), which

43 causes disease in cattle only (see Chapter 3.4.12), and sheeppox virus (SPPV) and goatpox virus (GT~~P~~PV), which cause

44 sheep pox and goat pox, respectively. Sheep pox and goat pox are characterised by disseminated cutaneous nodules and

45 up to 100% mortality in fully susceptible breeds of sheep and goats. In indigenous animals, generalised disease and

46 mortality are less common, although they are seen where disease has been absent from an area or village for a period of

47 time, when intensive husbandry methods are introduced, or in association with other disease agents, such as peste des

48 petits ruminants virus or foot and mouth disease virus. Sheep pox and goat pox are major constraints to the introduction

49 of exotic breeds of sheep and goats to endemic areas, and to the development of intensive livestock production.

50 Strains of SPPV and GTPV can pass between sheep and goats, although most cause more severe clinical disease in ~~only~~

51 ~~one~~their homologous host species. SPPV and GTPV are transboundary diseases that regularly spread into adjacent, non-

52 endemic areas. Sheep pox and goat pox are endemic in Africa north of the Equator and parts of the Middle East and Asia

53 (see WAHIS for most up-to-date information on distribution: https://wahis.woah.org/#/home). Outbreaks have been

54 reported in non-endemic countries of Asia, Europe and the Middle East.

55 The incubation period of sheep pox and goat pox is between 8 and 13 days following contact between infected and

56 susceptible animals. It may be as short as 4 days following experimental infection by intradermal inoculation~~or mechanica~~l

57 ~~transmission by insects~~. Some breeds of European sheep, such as Soay, may die of acute infection before the

58 development of skin lesions. In other breeds there is an initial rise in rectal temperature to above 40°C, followed in 2–5

59 days by the development of, at first, macules – small circumscribed areas of hyperaemia, which are most obvious on

60 unpigmented skin – and then of papules – hard swellings of between 0.5 and 1 cm in diameter – which may cover the body

61 or be restricted to the groin, axilla and perineum. Papules may be covered by fluid-filled vesicles, but this is rare. Some

62 researchers have distinguished between a vesicular and nodular form of sheep pox and goat pox (Zro *et al*., 2014b).

63 Within 24 hours of the appearance of generalised papules, affected animals develop rhinitis, conjunctivitis and enlargement

64 of all the superficial lymph nodes, in particular the prescapular lymph nodes. Papules on the eyelids cause blepharitis of

65 varying severity. As the papules on the mucous membranes of the eyes and nose ulcerate, so the discharge becomes

66 mucopurulent, and the mucosae of the mouth, anus, and prepuce or vagina become necrotic. Breathing may become

67 laboured and noisy due to pressure on the upper respiratory tract from the swollen retropharyngeal lymph nodes, due to

68 the developing lung lesions.

69 If the affected animal does not die in this acute phase of the disease, the papules start to become necrotic from ischaemic

70 necrosis  following  thrombi  formation  in  the  blood  vessels  at  the  base  of  the  papule.  In  the  following

71 5–10 days the papules form scabs, which persist for up to 6 weeks, leaving small scars. The skin lesions are susceptible

72 to fly strike, and secondary pneumonia is common. Anorexia is not usual unless the mouth lesions physically interfere with

73 feeding. Abortion is rare.

74 On post-mortem examination of the acutely infected animal, the skin lesions are often less obvious than on the live animal.

75 The mucous membranes appear necrotic and all the body lymph nodes are enlarged and oedematous. Papules, which

76 may be ulcerated, can usually be found on the abomasal mucosa, and sometimes on the wall of the rumen and large

77 intestine, on the tongue, hard and soft palate, trachea and oesophagus. Pale areas of approximately 2 cm in diameter may

78 occasionally be seen on the surface of the kidney and liver, and have been reported to be present in the testicles. Numerous

79 hard lesions of up to 5 cm in diameter are commonly observed throughout the lungs, but particularly in the diaphragmatic

80 lobes.

81 The clinical signs and post-mortem lesions vary considerably with breed of host and strain of capripoxvirus. Indigenous

82 breeds are less susceptible and frequently show only a few lesions, which could be confused with insect bites or contagious

83 pustular dermatitis. However, lambs that have lost their maternally derived immunity, animals that have been kept isolated

84 and animals brought into endemic areas from isolated villages, particularly if they have been subjected to the stress of

85 moving long distances and mixing with other sheep and goats, and their pathogens, can often be seen with generalised

86 and sometimes fatal capripoxvirus infections. Invariably there is high mortality in unprotected imported breeds of sheep

87 and goats following capripoxvirus infection. Capripoxvirus is not infectious to humans.

## 88	B. DIAGNOSTIC TECHNIQUES

89 ***Table 1.****Test methods available for diagnosis of sheep pox and goat pox and their purpose*

90 Key: +++ = recommended for this purpose; ++ recommended but has limitations;

91	+ = suitable in very limited circumstances; – = not appropriate for this purpose.

92 IFAT = indirect fluorescent antibody test; IHC = ; immunohistochemistry; PCR = polymerase chain reaction;

93 VNT = virus neutralisation; ELISA = enzyme-linked immunosorbent assay.

94 (a)~~A combination of agent identification methods applied on the same clinical sample is recommended.~~

#### 95 1.	Identification of the agent

##### 96 1.1.	Specimen collection and submission

97 Material for virus isolation and antigen detection should be collected by biopsy or at post-mortem from skin papules,

98 lung lesions or lymph nodes. Samples for virus isolation~~and antigen-detection enzyme-linked immunosorbent assay~~

99 ~~(ELISA)~~ should be collected within the first week of the occurrence of clinical signs, before the development of

100 neutralising antibodies. Samples for genome detection by polymerase chain reaction (PCR) may be collected before

101 or after the development of neutralising antibody responses. Buffy coat from blood collected into EDTA (ethylene

102 diamine tetra-acetic acid) during the viraemic stage of capripoxvirus infection (before generalisation of lesions or

103 within 4 days of generalisation), can also be used for virus isolation.

104 Samples for histology should include tissue from the surrounding area and should be placed immediately following

105 collection into ten times the sample volume of 10% formalin or neutral buffered 10% formal saline. Tissues in formalin

106 have no special transportation requirements.

107 Blood samples, for virus isolation from the buffy coat, should be collected in tubes containing anticoagulant, placed

108 immediately on ice and processed as soon as possible. In practice, the blood samples may be kept at 4°C for up to

109 2 days prior to processing, but should not be frozen or kept at ambient temperatures. Tissues and dry scabs for virus

110 isolation, antigen detection and genome detection should preferably be kept at 4°C, on ice or at –20°C. If it is

111 necessary to transport samples over long distances without refrigeration, the medium should contain 10% glycerol;

112 the samples should be of sufficient size that the transport medium does not penetrate the central part of the biopsy,

113 which should be used for virus isolation/detection.

##### 114 1.2.	Virus isolation

115 Lesion material for virus isolation and antigen detection is homogenised. The following is an example of one technique

116 for homogenisation: The tissue is minced using sterile scissors and forceps, and then macerated in a steel ball bearing

117 mixer mill or ground with a sterile pestle in a mortar with sterile sand and an equal volume of sterile phosphate

118 buffered saline (PBS) or serum-free Modified Eagle’s Medium (MEM) containing sodium penicillin (1000 international

119 units [IU]/ml), streptomycin sulphate (1 mg/ml), mycostatin (100 IU/ml) or fungizone (2.5 μg/ml) and neomycin

120 (200 IU/ml). The homogenised suspension is freeze–thawed three times and then partially clarified by centrifugation

121 using a bench centrifuge at 600 ***g***for 10 minutes. In cases where bacterial contamination of the sample is expected

122 (such as when virus is isolated from skin samples), the supernatant can be filtered through a 0.45 μm pore size filter

123 after the centrifugation step, however, the amount of virus in the supernatant might be reduced. Buffy coats may be

124 prepared from 5–8 ml unclotted blood by centrifugation at 600 ***g***for 15 minutes; the buffy coat is carefully removed

125 into 5 ml of cold double-distilled water using a sterile Pasteur pipette. After 30 seconds, 5 ml of cold double-strength

126 growth medium is added and mixed. The mixture is centrifuged at 600 ***g***for 15 minutes, the supernatant is discarded

127 and the cell pellet is suspended in 5 ml of growth medium, such as Glasgow’s modified Eagle’s medium (GMEM).

128 After centrifugation at 600 ***g***for a further 15 minutes, the resulting pellet is suspended in 5 ml of fresh GMEM.

129 Alternatively, the buffy coat may be separated from a heparinised sample using a density gradient.

130 Capripoxvirus will grow in tissue culture of bovine, ovine or caprine origin, although primary or secondary cultures of

131 lamb testis (LT) or lamb kidney (LK) cells are considered to be the most susceptible. Care needs to be taken to ensure

132 they are not contaminated with viruses such as bovine viral diarrhoea virus~~, particularly those derived from a woo~~l

133 ~~sheep breed~~ (see chapter 1.1.9). Madin–Darby bovine kidney (MDBK) cells have been shown to be suitable for

134 capripoxvirus isolation (Fay *et al.,*2020). The following is an example of an isolation technique: either 1 ml of buffy

135 coat cell suspension or 1 ml of clarified biopsy preparation supernatant is inoculated on to a 25 cm2 tissue culture

136 flask of appropriate cells at 90% ~~confluent LT or LK cells~~confluence, and the supernatant is allowed to adsorb for 1

137 hour at 37°C. The culture is then washed with warm PBS and covered with 10 ml of a suitable medium, such as

138 GMEM, containing antibiotics and 2% fetal calf serum. If available, tissue culture tubes~~containing LT or LK cells and~~

139 ~~a~~, flying cover-slips, or tissue culture microscope slides, ~~are~~can also infected.

140 The flasks should be examined daily for 7–14 days for evidence of cytopathic effect (CPE). Contaminated flasks

141 should be discarded. Infected cells develop a characteristic CPE consisting of retraction of the cell membrane from

142 surrounding cells, and eventually rounding of cells and margination of the nuclear chromatin. At first only small areas

143 of CPE can be seen, sometimes as soon as 4 days after infection; over the following 4–6 days these expand to

144 involve the whole cell sheet. If no CPE is apparent by day 7, the culture should be freeze–thawed three times, and

145 clarified supernatant inoculated on to fresh ~~LT or LK~~cell cultures. At the first sign of CPE in the flasks, or earlier if a

146 number of infected cover-slips are being used, a cover-slip should be removed, fixed in acetone and stained using

147 H&E. Eosinophilic intracytoplasmic inclusion bodies, which are variable in size but up to half the size of the nucleus

148 and surrounded by a clear halo, are indicative of poxvirus infection. Syncytia formation is not a feature of capripoxvirus

149 infection. If the CPE is due to capripoxvirus infection of the cell culture, it can be prevented or delayed by inclusion of

150 specific anti-capripoxvirus serum in the medium; this provides a presumptive identification of the agent. Some strains

151 of capripoxvirus have been adapted to grow on African green monkey kidney (Vero) cells, but these cells are not

152 recommended for primary isolation.

##### 153 1.3.	Electron microscopy

154 The characteristic poxvirus virion can be visualised using a negative-staining preparation technique followed by

155 examination with an electron microscope. There are many different negative-staining protocols, an example is given

156 below:

157 Material from the original tissue suspension is prepared for transmission electron microscope examination, prior to

158 centrifugation, by floating a 400-mesh hexagon electron microscope grid, with piloform-carbon substrate activated by

159 glow discharge in pentylamine vapour, on to a drop of the suspension placed on parafilm or a wax plate. After 1

160 minute, the grid is transferred to a drop of Tris/EDTA buffer, pH 7.8, for 20 seconds and then to a drop of 1%

161 phosphotungstic acid, pH 7.2, for 10 seconds. The grid is drained using filter paper, air-dried and placed in the

162 electron microscope. The capripoxvirus virion is brick shaped, covered in short tubular elements and measures

163 approximately 290 × 270 nm. A host-cell-derived membrane may surround some of the virions, and as many as

164 possible should be examined to confirm their appearance (Kitching & Smale, 1986).

165 The virions of capripoxvirus are indistinguishable from those of orthopoxvirus, but, apart from *Vaccinia*virus, no

166 orthopoxvirus causes lesions in sheep and goats. However, capripoxvirus is distinguishable from the virions of

167 parapoxvirus, that cause contagious pustular dermatitis, as they are smaller, oval in shape, and each is covered in a

168 single continuous tubular element, which appears as striations over the virion.

##### 169 1.4.	Histopathology

170 Material for histopathology should be prepared by standard techniques. Following preparation, staining with

171 haematoxylin and eosin (H&E), and mounting of the formalin-fixed biopsy material, a number of sections should be

172 examined by light microscopy. On histological examination, the most striking aspects of acute-stage skin lesions are

173 a massive cellular infiltrate, vasculitis and oedema. Early lesions are characterised by marked perivascular cuffing.

174 Initially infiltration is by macrophages, neutrophils and occasionally eosinophils, and as the lesion progresses, by

175 more macrophages, lymphocytes and plasma cells. A characteristic feature of all capripoxvirus infections is the

176 presence of variable numbers of ‘sheep pox cells’ in the dermis. These sheep pox cells can also occur in other organs

177 where microscopic lesions of sheep and goat pox are present. These cells are large, stellate cells with eosinophilic,

178 poorly defined intracytoplasmic inclusions and vacuolated nuclei. Vasculitis is accompanied by thrombosis and

179 infarction, causing oedema and necrosis. Epidermal changes consist of acanthosis, parakeratosis and

180 hyperkeratosis. Changes in other organs are similar, with a predominant cellular infiltration and vasculitis. Lesions in

181 the upper respiratory tract are characterised by ulceration.

##### 182 1.5.	Immunological methods

###### 183 1.5.1.	Fluorescent antibody tests

184 Capripoxvirus antigen can also be identified on infected cover-slips or tissue culture slides using fluorescent

185 antibody tests. Cover-slips or slides should be washed and air-dried and fixed in cold acetone for 10 minutes.

186 The indirect test using immune sheep or goat sera is subject to high background colour and nonspecific

187 reactions. However, a direct conjugate can be prepared from sera from convalescent sheep or goats or from

188 rabbits hyperimmunised with purified *Capripoxvirus*. Uninfected tissue culture should be included as a

189 negative control because cross-reactions, due to antibodies to cell culture antigens, can cause problems. The

190 fluorescent antibody tissue section technique has also been used on cryostat-prepared slides.

##### 191 1.6.	Nucleic acid recognition methods

192 Amplification methods for detection of ~~the viral DNA genome are specific to the genus~~*Capripoxvirus*DNA are ~~and~~

193 both specific and sensitive ~~for detection~~throughout the course of disease, including before and after the emergence

194 of antibody responses. These methods include conventional PCR, real-time PCR, and most recently loop-mediated

195 isothermal amplification (LAMP). Nucleic acid recognition methods can be used to detect the *Capripoxvirus*genome

196 in biopsy, swab or tissue culture samples.

###### 197 1.6.1.	Conventional PCR methods

198 Several conventional PCR methods have been reported with varying specificity for capripoxviruses

199 in general, SPPV, or GTPV (Heine *et al.,*1999; Ireland & Binepal, 1998; Zro *et al.,*2014a). A

200 conventional PCR assay that differentiates GTPV and LSDV from SPPV has been described (Lamien

201 *et al.,*2011a). Conventional PCR methods are particularly useful for obtaining sufficient genetic

202 material necessary for species identification by subsequent sequence and phylogenetic analysis (Le

203 Goff *et al.,*2009).

204 The conventional gel-based PCR method described below is a simple, fast and sensitive method for

205 the detection of capripoxvirus genome in EDTA blood, semen or tissue culture samples (Tuppurainen

206 *et al.,*2005).

207 Test procedure

208 The extraction method described below can be replaced using commercially available DNA extraction

209 kits.

210 i)	Freeze and thaw 200 μl of blood in EDTA, semen or tissue culture supernatant and suspend in

211 100 μl of lysis buffer containing 5 M guanidine thiocyanate, 50 mM potassium chloride, 10 mM

212 Tris/HCl (pH 8); and 0.5 ml Tween 20.

213 ii)	Cut skin and other tissue samples into fine pieces using a sterile scalpel blade and forceps.

214 Grind with a pestle in a mortar. Suspend the tissue samples in 800 μl of the above mentioned

215 lysis buffer.

216 iii)	Add 2 μl of proteinase K (20 mg/ml) to blood samples and 10 μl of proteinase K (20 mg/ml) to

217 tissue samples. Incubate at 56°C for 2 hours or overnight, followed by heating at 100°C for

218 10 minutes. Add phenol:chloroform:isoamylalcohol (25:24:1 [v/v]) to the samples in a 1:1 ratio.

219 Vortex and incubate at room temperature for 10 minutes. Centrifuge the samples at 16,060 ***g***

220 for 15 minutes at 4°C. Carefully collect the upper, aqueous phase (up to 200 μl) and transfer

221 into a clean 2.0 ml tube. Add two volumes of ice cold ethanol (100%) and 1/10 volume of 3 M

222 sodium acetate (pH 5.3). Place the samples at –20°C for 1 hour. Centrifuge again at 16,060 ***g***

223 for 15 minutes at 4°C and discard the supernatant. Wash the pellets with ice cold 70% ethanol

224 (100 μl) and centrifuge at 16,060 ***g***for 1 minute at 4°C. Discard the supernatant and dry the

225 pellets thoroughly. Suspend the pellets in 30 μl of nuclease-free water and store immediately at

226 –20°C (Tuppurainen *et al*., 2005). Alternatively a column-based extraction kit may be used.

227 iv)	The primers for this PCR assay were developed from the gene encoding the viral attachment

228 protein. The size of the expected amplicon is 192 bp (Ireland & Binepal, 1998). The primers

229 have the following gene sequences:

230 Forward primer 5’-TCC-GAG-CTC-TTT-CCT-GAT-TTT-TCT-TAC-TAT-3’

231 Reverse primer 5’-TAT-GGT-ACC-TAA-ATT-ATA-TAC-GTA-AAT-AAC-3’.

232 v) DNA amplification is carried out in a final volume of 50 μl containing: 5 μl of 10 × PCR buffer,

233 1.5 μl of MgCl2 (50 mM), 1 μl of dNTP (10 mM), 1 μl of forward primer, 1 μl of reverse primer,

234 1 μl of DNA template (~10 ng), 0.5 μl of *Taq*DNA polymerase and 39 μl of nuclease-free water.

235 The volume of DNA template required may vary and the volume of nuclease-free water must

236 be adjusted to the final volume of 50 μl.

237 vi) Run the samples in a thermal cycler as follows: 2 minutes at 95°C; then 45 seconds at 95°C,

238 50 seconds at 50°C and 1 minute at 72°C (34 cycles); 2 minutes at 72°C and hold at 4°C until

239 analysis.

240 vii) Mix 10 μl of each sample with loading dye and load onto a 1.5% agarose gel in TAE buffer

241 (Tris/acetate buffer containing EDTA). Load a parallel lane with a 100 bp DNA-marker ladder.

242 Electrophoretically separate the products using approximately 8–10 V/cm for 40–60 minutes

243 and visualise with a suitable DNA stain and transilluminator.

###### 244 1.6.2.	Real-time PCR methods

245 Several highly sensitive and specific fluorescent detection-based real-time PCR methods have been

246 developed and validated (Balinsky *et al*., 2008; Bowden *et al.,*2008; Das *et al.,*2012; Stubbs *et al.,*

247 2012). Each test detects a small conserved genetic locus within the capripoxvirus genome, but these

248 methods do not discriminate between SPPV, GTPV or LSDV. Real-time PCR methods for direct

249 capripoxvirus~~genotyping~~species differentiation without the need for gene sequencing have been

250 described (Gelaye *et al.,*2013; Lamien *et al.,*2011b; Wolff *et al.,*2021).

251 The real-time PCR method described below is a rapid, sensitive and specific method for the detection

252 of DNA from SPPV, GTPV or LSDV. This assay will not differentiate between capripoxvirus species.

253 **DNA extraction from blood and tissue**

254 A number of DNA extraction kits are commercially available for the isolation of template DNA for real-

255 time PCR. Manufacturer’s instructions should always be consulted for guidance on the appropriate

256 method for the sample type being extracted. WOAH Reference Laboratories can be contacted for

257 advice on suitable commercial kits.

258 **Real-time PCR**

259 i)	The real-time PCR method outlined below uses the primers and probe described by Bowden *et*

260 *al*. (2008). and further validated by Stubbs *et al.*(2012). Cycling conditions and reagent

261 concentrations can be altered to ensure optimal performance in individual laboratories.

262 ii)	Forward and reverse primers should be prepared at concentrations of 20 μM. A minor grove

263 binder (MGB) TaqMan probe should be prepared at a concentration of 10 μM.

264 iii)	Forward primer: 5’-AAA-ACG-GTA-TAT-GGA-ATA-GAG-TTG-GAA-3’

265 iv)	Reverse primer: 5’-AAA-TGA-AAC-CAA-TGG-ATG-GGA-TA-3’

266 v)	Probe: 5’-FAM-TGG-CTC-ATA-GAT-TTC-CT-MGB-3’

267 vi)	Mastermix is prepared by combining 10 μl of 2 × real-time PCR mastermix with 0.4 μl of forward

268 primer, 0.4 μl of reverse primer, 0.5 μl of probe and 6.7 μl of RNase free water per reaction.

269 vii)  Add 2 μl of extracted DNA to 18 μl of mastermix in a 96-well PCR plate or PCR strip and perform

270 real-time PCR according to the example given below or similar method:

271 viii) 95°C for 10 minutes followed by 45 cycles of 95°C for 15 seconds, 60°C for 60 seconds.

272 Fluorescence detection should be performed at the end of each cycle.

273 ix)	Following completion of the real-time PCR, a cycle threshold (CT) should be set. Samples with

274 CT values less than 35 are considered positive. Samples with a CT value greater than 35 but

275 less than 45 are considered inconclusive and require further investigation. Samples which do

276 not yield a CT value, i.e. the amplification curve does not cross the threshold, are considered

277 negative.

###### 278 1.6.3.	Isothermal genome amplification

279 Molecular tests using loop-mediated isothermal amplification (LAMP) to detect capripoxvirus

280 genomes are reported to provide sensitivity and specificity similar to real-time PCR with a simpler

281 method and at lower cost (Das *et al.,*2012; Murray *et al.,*2013). Field validation of the Das *et al.*

282 (2012) LAMP method has been further reported by (Omoga *et al.,*2016) and a combination of this

283 universal capripoxvirus test with two additional LAMP assays was reported to show utility in

284 discriminating between GTPV and SPPV (Zhao *et al.,*2014).

#### 285 2.	Serological tests

##### 286 2.1.	Virus neutralisation

287 A test serum can either be titrated against a constant titre of capripoxvirus (100 TCID50 [50% tissue culture infective

288 dose]) or a standard capripoxvirus strain can be titrated against a constant dilution of test serum in order to calculate

289 a neutralisation index. Because of the variable sensitivity of tissue culture to capripoxvirus, and the consequent

290 difficulty of ensuring the use of 100 TCID50, the neutralisation index is the preferred method, although it does require

291 a larger volume of test sera. The test is described using 96-well flat-bottomed tissue culture grade microtitre plates,

292 but it can be performed equally well in tissue culture tubes with the appropriate changes to the volumes used, although

293 it is more difficult to read an end-point in tubes. ~~The use of Vero cells in the virus neutralisation test has been reported~~

294 ~~to give more consistent results (Kitching & Taylor, 1985).~~

###### 295 2.1.1.	Test procedure

296 i)	Test sera including a negative and a positive control are diluted 1/5 in Eagle’s/HEPES (N-2-

297 hydroxyethylpiperazine, N-2-ethanesulphonic acid) and inactivated at 56°C for 30 minutes.

298 ii)	Next, 50 μl of the first inactivated serum is added to columns 1 and 2, rows A to H of the

299 microtitre plate. The second serum is placed in columns 3 and 4, the third in columns 5 and 6,

300 the positive control serum is placed in columns 7 and 8, the negative control serum is placed in

301 columns 9 and 10, and 50 μl of Eagle’s/HEPES without serum is placed in columns 11 and 12

302 and to all wells of row H.

303 iii)	A reference strain of capripoxvirus, usually a vaccine strain known to grow well in tissue culture,

304 with a titre of over log10 6 TCID50 per ml is diluted in Eagle’s/HEPES in bijoux bottles to give a

305 log dilution series of log10 5.0; 4.0; 3.5; 3.0; 2.5; 2.0; 1.5 TCID50 per ml (equivalent to log10 3.7;

306 2.7; 2.2; 1.7; 1.2; 0.7; 0.2 TCID50 per 50 μl).

307 iv)	Starting with row G and the most diluted virus preparation, 50 μl of virus is added to each well

308 in that row. This is repeated with each virus dilution, the highest titre virus dilution being placed

309 in row A.

310 v)	The plates are covered and incubated for 1 hour at 37°C.

311 vi)	~~LT cells are~~An appropriate cell suspension (such as MDBK cells)is prepared from pregrown

312 monolayers as a suspension of 105 cells/ml in Eagle’s medium containing antibiotics and 2%

313 fetal calf serum. Following incubation of the microtitre plates, 100 μl of cell suspension is added

314 to all the wells, except wells H11 and H12, which serve as control wells for the medium. The

315 remaining wells of row H are cell and serum toxicity controls.

316 vii)  The microtitre plates are covered and incubated at 37°C for 9 days.

317 viii) Using an inverted microscope, the monolayers are examined daily starting at day 4 for evidence

318 of CPE. There should be no CPE in the cells of row H. Using the 0240 KSGP vaccine strain of

319 capripoxvirus, the final reading is taken on day 9, and the titre of virus in each duplicate titration

320 is calculated according to the Kärber method. If left longer, there is invariably a ‘breakthrough’

321 of virus in which virus that was at first neutralised appears to disassociate from the antibody.

322 ix)	*Interpretation of the results:*The neutralisation index is the log titre difference between the titre

323 of the virus in the negative serum and in the test serum. An index of ≥1.5 is positive. The test

324 can be made more sensitive if serum from the same animal is examined before and after

325 infection. Because immunity to capripoxvirus is predominantly cell mediated, a negative result,

326 particularly following vaccination in which the response is necessarily mild, does not imply that

327 the animal from which the serum was taken is not protected.

328 ~~A constant-virus/varying-serum method has been described using serum dilutions in the range~~

329 ~~1/5 to 1/500 and fetal calf muscle cells. Because these cells have a lower sensitivity to~~

330 ~~capripoxvirus than LT cells, the problem of virus ‘breakthrough’ is overcome.~~

##### 331 2.2.	Indirect fluorescent antibody test

332 Capripoxvirus-infected tissue culture grown on flying cover-slips or tissue culture microscope slides can be used for

333 the indirect fluorescent antibody test. Uninfected tissue culture control, and positive and negative control sera, should

334 be included in the test. The infected and control cultures are fixed in acetone at –20°C for 10 minutes and stored at

335 4°C. Dilutions of test sera are made in PBS, starting at 1/5, and positives are identified using an anti-sheep gamma-

336 globulin conjugated with fluorescein isothiocyanate (Davies & Otema, 1978). Cross-reactions can occur with orf,

337 bovine papular stomatitis virus and perhaps other poxviruses.

##### 338 2.3.	Western blot analysis

339 Western blotting of test sera against capripoxvirus-infected cell lysate provides a sensitive and specific system for

340 the detection of antibody to capripoxvirus structural proteins, although the test is expensive and difficult to carry out

341 (Chand *et al.*, 1994).

##### 342 2.4.	Enzyme-linked immunosorbent assay

343 ~~No validated ELISA is available for the serological diagnosis of SPP or GTP.~~

344 Both in-house and commercial enzyme-linked immunosorbent assay (ELISAs) are available, but these tests cannot

345 discriminate between antibodies to different capripoxviruses (LSD or SPP/GTP).

## 346	C. REQUIREMENTS FOR VACCINES

347	**[THIS SECTION IS UNDER REVIEW IN THE 2024/2025 REVIEW CYCLE]**

#### 348 1.	Background

##### 349 1.1.	Rationale and intended use of the product

350 A variety of attenuated live and inactivated capripoxvirus vaccines has been used to provide protection against

351 sheeppox and goatpox. All strains of capripoxvirus of ovine, caprine or bovine origin examined so far share a major

352 neutralising site, so that animals recovered from infection with one strain are resistant to infection with any other strain

353 (Capstick, 1961). Consequently, it is possible to use a single strain of capripoxvirus to protect both sheep and goats

354 against all field strains of virus, regardless of whether their origin was in Asia or Africa (Kitching *et al.*, 1986; Kitching

355 & Taylor, 1985). However, field evidence suggests some strains are quite host-specific and are used only in sheep

356 against SPPV and only in goat against GTPV.

357 A number of strains of capripoxvirus have had widespread use as live vaccines (Davies & Mbugwa, 1985), for

358 example the Romanian and RM-65 strains used mainly in sheep and the Mysore and Gorgan strains used in goats.

359 The real identity of the commonly used Kenyan sheep and goat pox vaccine virus (KSGP) 0240 was recently shown

360 to be actually LSDV (Tuppurainen *et al.,*2014). Virus strain identity and attenuation properties must be ascertained

361 and taken into consideration when selecting vaccine strains for use in cattle, sheep and goats. The protective dose

362 depends on the vaccine strain used. Immunity in sheep and goats against capripoxvirus following vaccination with

363 the 0240 strain lasts over a year and the Romanian strain gave protection for at least 30 months.

364 Killed vaccines produced from tissue culture contain only the intracellular mature virion form of the virus, and lack the

365 less robust but biologically crucial extracellular enveloped virion form. As a result, the vaccine does not stimulate

366 immunity against the extracellular enveloped virion, resulting in poor protection. Killed capripoxvirus vaccines provide,

367 at best, only temporary protection.

#### 368 2.	Outline of production and minimum requirements for conventional vaccines

369 General requirements set for the facilities used for the production of vaccines and for the documentation and record keeping

370 throughout the whole manufacturing process are described in Chapter 1.1.8 *Principles of veterinary vaccine production*.

371 The documentation should include the standard operating procedures (SOP) for the method of manufacture and each step

372 for the testing of cells and reagents used in the process, each batches and the final product.

##### 373 2.1.	Characteristics of the seed

###### 374 2.1.1.	Biological characteristics

375 A strain of capripoxvirus used for vaccine production must be accompanied by a history describing

376 its origin and tissue culture or animal passage. It must be safe to use in all breeds of sheep and goats

377 for which it is intended, including pregnant and young animals. It must be non-transmissible, remain

378 attenuated after further tissue culture passage, and provide complete protection against challenge

379 with virulent field strains for a minimum of 1 year. A quantity of master seed vaccine virus should be

380 prepared and stored in order to provide a consistent working seed for regular vaccine production.

###### 381 2.1.2.	Quality criteria (sterility, purity, freedom from extraneous agents)

382 Each master seed must be tested to ensure its identity and shown to be free from adventitious

383 viruses, in particular pestiviruses, such as border disease and bovine viral diarrhoea virus, and free

384 from contamination with bacteria, fungi and/or mycoplasmas. The general procedures for sterility or

385 purity tests are described in chapter 1.1.9. The master seed must also be safe and produce no clinical

386 reaction in all breeds of sheep or goats when given by the recommended route and stimulate

387 complete immunity to capripoxvirus in all breeds of sheep and goats for at least 1 year. The necessary

388 safety and potency tests are described in Section C.2.2.4 *Final product batch tests*.

##### 389 2.2.	Method of manufacture

390 The method of manufacture should be documented as the Outline of Production.

###### 391 2.2.1.	Procedure

392 Vaccine seed should be lyophilised and stored in 2 ml vials at –20°C. It may be stored wet at –20°C,

393 but when wet, is more stable at –70°C or lower. The virus should be cultured in primary or secondary

394 LT or LK cells of wool sheep origin for maximum yield. Vero cells may also be used with suitably

395 adapted strains.

396 Vaccine batches are produced on fresh monolayers of secondary LT or primary LK cells. A vial of

397 seed virus is reconstituted with GMEM or another appropriate medium and inoculated on to an LT or

398 LK monolayer that has been previously washed with warm PBS, and allowed to adsorb for 15 minutes

399 at 37°C before being overlaid with additional GMEM. After 4–6 days, there will be extensive (80–

400 90%) CPE. The culture should be examined for any evidence of nonspecific CPE, medium cloudiness

401 or change in medium pH. The culture is freeze–thawed three times, the suspension removed and

402 centrifuged at 600 ***g***for 20 minutes. A second passage may be required to produce sufficient virus

403 for a production batch. Live vaccine may be produced on roller bottles.

404 The procedure is repeated and the harvests from individually numbered flasks are each mixed

405 separately with an equal volume of sterile and chilled 5% lactalbumin hydrolysate and 10% sucrose,

406 and transferred to individually numbered bottles for storage at –20°C. Prior to storage, 0.2 ml is

407 removed from each bottle for sterility control. An additional 0.2 ml is removed for virus titration; 2 ml

408 pools composed of 0.2 ml samples taken from ten bottles are used. A written record of all the

409 procedures must be kept for all vaccine batches.

410 Inactivated vaccines are produced, usually from unattenuated field strains of capripoxvirus, grown in

411 tissue culture as described above, inactivated with 0.03% formaldehyde, and mixed with an equal

412 volume of alhydrogel as adjuvant. Formaldehyde is no longer considered to be a suitable inactivant

413 for certain viral vaccines because its mode of action cannot be guaranteed to be totally effective in

414 inactivating all the live virus. This has not been fully investigated for capripoxvirus.

###### 415 2.2.2.	Requirements for substrate and media

416 The specification and source of all ingredients used in the manufacturing procedure should be

417 documented and the freedom from extraneous agents: bacteria, fungi, mycoplasma and any other

418 viruses should be tested. The detailed testing procedure is described in the chapter 1.1.9. The use

419 of antibiotics must meet the requirements of the licensing authority.

###### 420 2.2.3.	In-process controls

421 i)  Cells

422 Cells should be obtained from the testis or kidney of a healthy young lamb from a scrapie-free flock

423 of a wool sheep breed. During cultivation, cells must be observed for any evidence of CPE, and for

424 normal morphology (predominantly fibroblastic). They can usually be passaged successfully up to

425 ten times. When used for vaccine production, uninfected control cultures should be grown in parallel

426 and maintained for at least three additional passages for further observation. They should be checked

427 for the presence of noncytopathic strains of bovine virus diarrhoea or border disease viruses by

428 immunofluorescence or immunoperoxidase techniques. If possible, cells should be prepared and

429 screened prior to vaccine production and stocked in 1–2 ml aliquots containing 2 × 107 cells/ml in

430 sterile 10% DMSO (dimethyl sulphoxide) and 90% FBS (fetal bovine serum) solution stored in liquid

431 nitrogen.

432 ii) Serum

433 Bovine serum used in the growth or maintenance medium must be free from transmissible spongiform

434 encephalopathies (TSEs) and antibody to capripoxvirus, and tested for contamination with pestivirus

435 or any other viruses, extraneous bacteria, mycoplasma or fungi.

436 iii) Medium

437 Medium must be tested free from contamination with pestivirus or any other viruses, extraneous

438 bacteria, mycoplasma or fungi.

439 iv) Virus

440 Seed virus and final vaccine must be titrated in tissue culture tubes or microtitre plates. Vaccine

441 samples must be examined for the presence of adventitious viruses including cytopathic and

442 noncytopathic strains of pestivirus, and should be mixed with a high-titre capripoxvirus-immune

443 serum that has tested negative for antibody to pestivirus to prevent the vaccine virus itself interfering

444 with the test. The vaccine bulk can be held at –20°C or below until all sterility tests and titrations have

445 been completed, at which time it should be freeze-dried in 1 ml aliquots in vials sufficient for 100

446 doses. The vaccine harvest diluted with lactalbumin hydrolysate and sucrose should have a minimum

447 titre log10 4.5 TCID50 per ml after freeze-drying, equivalent to a field dose of log10 2.5 TCID50. A further

448 titration is carried out on five randomly chosen vials of the freeze-dried preparation to confirm the

449 titre.

###### 450 2.2.4.	Final product batch tests

451 i)  Sterility/purity

452 Tests for sterility and freedom from contamination of biological materials intended for veterinary use

453 may be found in chapter 1.1.9.

454 ii) Safety

455 The safety studies should be demonstrated by statistically valid vaccination studies using

456 seronegative young sheep and goats of known susceptibility to capripox virus. The procedure

457 described is suitable for vaccine strains such as 0240 that are equally immunogenic in both sheep

458 and goats. The choice of target animal should be adapted for strains with a more restricted host

459 preference.

460 iii) Potency

461 Potency tests must be undertaken if the minimum immunising dose of the virus strain is not known.

462 This is usually carried out by comparing the titre of a virulent challenge virus on the flanks of

463 vaccinated and control animals. Following vaccination, the flanks of at least three animals and three

464 controls are shaved of wool or hair. Log10 dilutions of the challenge virus are prepared in sterile PBS

465 and six dilutions are inoculated intradermally (0.1 ml per inoculum) along the length of the flank; four

466 replicates of each dilution are inoculated down the flank. An oedematous swelling will develop at

467 possibly all 24 inoculation sites on the control animals, although preferably there will be little or no

468 reaction at the four sites of the most dilute inocula. The vaccinated animals should develop an initial

469 hypersensitivity reaction at sites of inoculation within 24 hours, which should quickly subside. Small

470 areas of necrosis may develop at the inoculation site of the most concentrated challenge virus. The

471 macule/papule is measured at between 8 and 10 days post-challenge. The titre of the challenge virus

472 is calculated for the vaccinated and control animals; a difference of log10 titre > 2.5 is taken as

473 evidence of protection.

##### 474 2.3.	Requirements for authorisation

###### 475 2.3.1.	Safety requirements

476 i)  Target and non-target animal safety

477 The vaccine must be safe to use in all breeds of sheep and goats for which it is intended, including

478 young and pregnant animals. It must also be non-transmissible, remain attenuated after further tissue

479 culture passage.

480 Safety tests should be carried out on the final product of each batch as described in Section C.2.2.4.

481 The safety of the vaccine in non-target animals must have been demonstrated using mice and

482 guinea-pigs as described in Section C.2.2.4. There should be no evidence of pathology caused by

483 the vaccine.

484 ii) Reversion-to-virulence for attenuated/live vaccines

485 The selected final vaccine should not revert to virulence during a further passages in target animals.

486 iii) Environmental consideration

487 Attenuated vaccine should not be able to perpetuate autonomously in cattle, sheep or goat

488 populations. Vaccines using the 0240 strain should not be used in *Bos taurus*breeds. Strains of

489 capripoxvirus are not a hazard to human health. There are no precautions other than those described

490 above for sterility and freedom from adventitious agents.

###### 491 2.3.2.	Efficacy requirements

492 i)  For animal production

493 The efficacy of the vaccine must be demonstrated in vaccination challenge experiment under

494 laboratory conditions. As described in Section C.2.2.4.

495 Once the potency of the particular strain being used for vaccine production has been determined in

496 terms of minimum dose required to provide immunity, it is not necessary to repeat this on the final

497 product of each batch, provided the titre of virus present has been ascertained.

498 ii) For control and eradication

499 Vaccination is the only effective way to control the sheep pox and goat pox outbreaks in endemic

500 countries. Unfortunately, currently no marker vaccines allowing the differentiation of infected from

501 vaccinated animals are available.

502 Immunity to virulent field virus following vaccination of sheep or goats with the 0240 strain lasts over

503 1 year, and protection against generalised infection following intradermal challenge lasts at least

504 3 years and is effective lifelong. The duration of immunity produced by other vaccine strains should

505 be ascertained in both sheep and goats by undertaking controlled trials in an environment in which

506 there is no possibility of field strains of capripoxvirus confusing the results. The inactivated vaccines

507 provide immunity for less than 1 year, and for the reasons given at the beginning of this section, may

508 not give immunity to the form of capripoxvirus usually associated with natural transmission.

###### 509 2.3.3.	Stability

510 All vaccines are initially given a shelf-life of 24 months before expiry. Real-time stability studies are

511 then conducted to confirm the appropriateness of the expiry date. Multiple batches of the vaccine

512 should be re-titrated periodically throughout the shelf-life to determine the vaccine variability.

513 Properly freeze-dried preparations of capripox vaccine, particularly those that include a protectant,

514 such as sucrose and lactalbumin hydrolysate, are stable for over 25 years when stored at –20°C and

515 for 2–4 years when stored at 4°C. There is evidence that they are stable at higher temperatures, but

516 no long-term controlled experiments have been reported. The inactivated vaccines must be stored at

517 4°C, and their shelf- life is usually given as 1 year.

518 No preservatives other than a protectant, such as sucrose and lactalbumin hydrolysate, are required

519 for the freeze-dried preparation.

#### 520 3.	Vaccines based on biotechnology

##### 521 3.1.	Vaccines available and their advantages

522 Currently, no recombinant vaccines for capripoxviruses are commercially available. However, a new generation of

523 capripox vaccines is being developed that uses the capripoxvirus genome as a vector for the genes of other ruminant

524 pathogens such as peste des petits ruminants (PPR) virus (Berhe *et al.*, 2003; Tuppurainen *et al*., 2014).

##### 525 3.2.	Special requirements for biotechological vaccines, if any

526 Not applicable.

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530 BERHE G., MINET C., LE GOFF C., BARRETT T., NGANGNOU A., GRILLET C., LIBEAU G., FLEMING M., BLACK D.N. & DIALLO A.

531 **(2003).**Development of a dual recombinant vaccine to protect small ruminants against peste-des-petits-ruminants virus

532 and capripoxvirus infections. *J. Virol*., **77**, 1571–1577.

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534 quantitative study in experimentally infected sheep and goats. *Virology*, **371**, 380–393.

535 CAPSTICK P.B. (1961). Annual Report. Kenya Veterinary Department, Kenya, 45–47.

536 CHAND P., KITCHING R.P. & BLACK D.N. (1994). Western blot analysis of virus-specific antibody responses to capripoxvirus

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539 detection of capripoxviruses. *J. Clin. Microbiol*., **50**, 1613–1620.

540 DAVIES F.G. & MBUGWA G. (1985). The alterations in pathogenicity and immunogenicity of a Kenya sheep and goat pox

541 virus on serial passage in bovine foetal muscle cell cultures. *J. Comp. Pathol*., **95**, 565–576.

542 DAVIES F.G. & OTEMA C. (1978). The antibody response in sheep infected with a Kenyan sheep and goat pox virus. *J.*

543 *Comp. Pathol*., **88**, 205–210.

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545 P.M. (2020). Madin–Darby bovine kidney (MDBK) cells are a suitable cell line for the propagation and study of the bovine

546 poxvirus lumpy skin disease virus. *J. Virol. Methods*, **285**, 113943. doi: 10.1016/j.jviromet.2020.113943.

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551 IRELAND D.C. & BINEPAL Y.S. (1998). Improved detection of capripoxvirus in biopsy samples by PCR. *J. Virol*. *Methods*, **74**,

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553 KITCHING R.P., HAMMOND J.M. & TAYLOR W.P. (1986). A single vaccine for the control of capripox infection in sheep and

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574 real-time polymerase chain reaction assay for the detection of capripoxviral DNA. *J. Virol. Methods*, **179**, 419–422.

575 TUPPURAINEN E.S.M., PEARSON C.R., BACHANEK-BANKOWSKA K., KNOWLES N.J., AMAREEN S., FROST L., HENSTOCK M.R.,

576 LAMIEN C.E., DIALLO A. & MERTENS P.P.C. (2014). Characterization of sheep pox virus vaccine for cattle against lumpy skin

577 disease virus. *Antiviral Res*., **109**, 1–6.

578 TUPPURAINEN E.S.M., VENTER E.H. & COETZER J.A.W. (2005). The detection of lumpy skin disease virus in samples of

579 experimentally infected cattle using different diagnostic techniques. *Onderstepoort J. Vet. Res.,***72**, 153–164.

580 WOLFF J.,& BEER M. & Hoffmann B. (2021). Probe-Based Real-Time qPCR Assays for a Reliable Differentiation of Capripox

581 Virus Species. *Microorganisms*, **9**, 765. https://doi.org/10.3390/microorganisms9040765

582 ZHAO Z., FAN B., WU G., YAN X., LI Y., ZHOU X., YUE H., DAI X., ZHU H., TIAN B., LI J. & ZHANG Q. (2014) Development of loop-

583 mediated isothermal amplification assay for specific and rapid detection of differential goat Pox virus and Sheep Pox virus.

584 *BMC Microbiol.,***14**, 1–10.

585 ZRO K., AZELMAT S., BENDOURO Y., KUHN J.H., EL FAHIME E. & ENNAJI M.M. (2014a). PCR-based assay to detect sheeppox

586 virus in ocular, nasal, and rectal swabs from infected Moroccan sheep. *J. Virol. Methods*, **204**, 38–43.

587 ZRO K., ZAKHAM F., MELLOUL M., EL FAHIME E. & MUSTAPHA M. (2014b). A sheeppox outbreak in Morocco: isolation and

588 identification of virus responsible for the new clinical form of disease. *BMC Vet Res*., **10**, 31.

589	*

590	*  *

591 **NB:**There are WOAH Reference Laboratories for sheep pox and goat pox (please consult the WOAH Web site:

592 https://www.woah.org/en/what-we-offer/expertise-network/reference-laboratories/#ui-id-3).

593 Please contact the WOAH Reference Laboratories for any further information on

594 diagnostic tests, reagents and vaccines for sheep pox and goat pox

595 **NB**: FIRST ADOPTED IN 1989. MOST RECENT UPDATES ADOPTED IN 2017.